Autochthonous infection with Ehrlichia Canis and Hepatozoon Canis in dogs from Serbia

Abstract Background The epidemiological status concerning many canine tick‐borne diseases (TBDs) in Serbia is still insufficiently known. Objectives Our study aimed to investigate the presence of tick‐borne pathogens of the family Anaplasmataceae and Hepatozoon spp., as a cause of illnesses accompanied by clinical signs that can occur in dogs with anaplasmosis, ehrlichiosis and hepatozoonosis. Methods Dogs are included in the study based on the presence of a minimum of three clinical and/or pathological findings that could be associated with anaplasmosis, ehrlichiosis and hepatozoonosis. During the study (April–October 2018), 11 dogs met the conditions to be included in the survey. Identification of the causative agent in the blood of diseased dogs was performed by conventional PCR followed by sequencing. Results The presence of the pathogens was confirmed in three animals (3/11, 27.3%). The presence of Ehrlichia canis was confirmed in 3‐month‐old female Rottweiler puppy, an 8‐year old Miniature Schnauzer female was positive for Hepatozoon canis infection, while 4‐year‐old mixed breed male dog was co‐infected with both mentioned pathogens. These are the first cases of autochthonous infection with E. canis and H. canis in dogs from Serbia confirmed by molecular methods. Conclusions The results of our study indicate the importance of molecular methods to establish a reliable diagnosis of TBDs. Also, the confirmed presence of causative agents of canine monocytic ehrlichiosis and hepatozoonosis in Serbia appeals to veterinary practitioners that it is necessary to exclude the presence of those diseases in suspicious dogs.


INTRODUCTION
Climate changes and expansion of the areal of vectors cause the appearance of vector-borne diseases in dogs with a higher incidence in regions where they have appeared sporadically. Also, the occurrence of tick-borne diseases (TBDs) caused by pathogens that were not previously confirmed in certain parts of Europe has nowadays increased (Semenza & Suk, 2018).
Recently, A. platys were confirmed as a causative agent of CCT in Serbia (Ilić Božović et al., 2021) while no clinical case of CGA and CME has been confirmed nor the DNA of the causative agent of these diseases have been detected in the blood of dogs in Serbia so far (Potkonjak et al., 2020). CME is a TBD of particular importance to the dog population in Southern Europe. The Gram-negative bacteria E. canis is the main causative agent, and structures called morulae could be seen in the cytoplasm of infected leukocytes. The Brown dog tick, Rhipicephalus sanguineus, is considered the main vector in Europe (Mathios & Konstantina, 2017), and it is also a species that often parasitises dogs in Serbia (Potkonjak et al., 2016). CME is characterised by multiple clinical manifestations and the disease has different phases, so it is difficult to achieve a reliable diagnosis in routine clinical practice when molecular diagnostic methods are not available. Further, other TBPs (A. phagocytophilum, A. platys, H. canis, Babesia canis) potentially present in co-infection, leading to an even more complex clinical presentation (Sainz et al., 2015). Nevertheless, transplacental transmission has also been confirmed.
In predisposed dogs, the infection can lead to a life-threatening illness although it is usually subclinical in immunocompetent animals. Fever, anaemia, lethargy, and cachexia are the most common symptoms in dogs with high parasitaemia (Baneth, 2011). There is little data on the epidemiological status of canine hepatozoonosis in Serbia. Recently, the DNA of H. canis is detected in the blood of one asymptomatic dog in southern Serbia (Gabrielli et al., 2015), while clinical cases of the disease in dogs have not been described in Serbia so far.
Our study aimed to investigate the presence of TBPs of the family Anaplasmataceae and Hepatozoon spp., as a cause of illnesses accompanied by clinical and/or pathological findings which are often present in canine anaplasmosis, ehrlichiosis, and hepatozoonosis.

MATERIALS AND METHODS
The study was conducted in 2018, during the main period of tick activ-

RESULTS
Eleven dogs met the conditions to be included in the study (present at least three clinical and/or pathological findings associated with canine anaplasmosis, ehrlichiosis and hepatozoonoses or a positive rapid anti-

Clinical presentations of dogs positive to Erlichia canis and Hepatozoon canis
A 3-month-old female Rottweiler puppy from the vicinity of Belgrade city without a travel history to the endemic area and vaccinated against infectious diseases was presented at an ordinary veterinary clinic with a high temperature (41.5 • C), apathy and enlarged retropharyngeal lymph nodes. No histories of previous diseases were reported. Twenty days before, the tick was removed from the puppy by the owner, so species determination was lacking. The dog was treated with antimicrobial agents-ceftriaxone and imidocarb dipropionate, and mineral and vitamin supplements (Mg, Fe, Cu, vitamin C, B6, B12, CoQ10, folic acid), without performing complete blood count (CBC) and other diagnostic tests. After nine days of therapy, the clinical condition worsened and the dog was referred to a hospital for small animals.
Re-evaluated clinical examination revealed lethargy, anorexia, pale mucosa membrane, prolonged capillary refill time (3 s), and generalised lymphadenopathy without high temperature (38.4 • C). CBC showed the presence of mild non-haemolytic, normocytic, normochromic, nonregenerative anaemia with mild neutropenia without leukocytosis and severe thrombocytopenia (Table 1). On blood and buffy coat smears granular lymphocytes were noted. Antibody point-of-care test (Bionote, Korea) indicated exposure to E. canis. Cytology of enlarged lymph nodes was not done. PCR assay followed by sequencing has con-   (Sainz et al., 2015). Previous research has confirmed that R. sanguineus most often infests dogs in our country as well (Bogunović et al., 2018;Potkonjak et al., 2016). Even though previous seroepidemiological studies conducted in Serbia have confirmed the exposure of dogs to E. canis (Bogićević et al., 2017;Potkonjak et al., 2013), clinical cases of CME or molecular confirmation of the causative agent have not been reported before (Kovačević Filipović et al., 2018). Also, the presence of E. canis in R. sanguineus ticks collected from dogs in Serbia has not been confirmed so far (Potkonjak et al., 2016). CME is characterised by three stages: acute, chronic and subclinical. Thrombocytopenia occurs in over 80% of all cases (Mathios & Konstantina, 2017), and our results fit this pattern. Both positive dogs in our study had thrombocytopenia. Although thrombocytopenia is present in most animals with CME, it occurs in many other vector-borne diseases and other haemostasis disorders indicated their important role not just in coagulation but also in inflammation. Severe pancytopenia as a consequence of bone marrow hypoplasia which is present in the chronic phase of CME can be fatal (Sainz et al., 2015).  (Tomanović et al., 2013b), a species that is at the same time most abundant on dogs in Serbia (Bogunović et al., 2018;Potkonjak et al., 2016).
Further studies are needed to elucidate the paths of transmission concerning H. canis in Serbia. So far, the DNA of H. canis is proven in the blood of one asymptomatic dog in southern Serbia (Gabrielli et al., 2015) while the parasite has not been confirmed in the competent tick vector so far (Potkonjak et al., 2016). In the present study, the clinical case of canine hepatozoonosis has been confirmed by conventional PCR followed by sequencing for the first time. It was not possible to notice H. canis gametocytes in the blood of the diseased dog at the onset of illness, probably due to a small number of infected blood cells and limited sensitivity of microscopic examination. In this particular case, the importance of molecular diagnostics in the early detection of H. canis infection is emphasised. Adequate therapy after the confirmed presence of H. canis by PCR gave results and led to the complete recovery of the diseased dog. Co-infection with two or more pathogens commonly complicates the clinical presentation of the diseases and special treatment protocol is needed in co-infected animals. A great advantage of PCR compared to other diagnostic methods is the ability to detect several pathogens simultaneously. In the present study, one dog was infected with E. canis and H. canis at the same time. This is not an unexpected finding since mentioned pathogens share the same competent tick vector and occasionally infect even the same blood cells (Baneth et al., 2015).

CONCLUSION
In the present study, for the first time in Serbia, autochthonous CME and canine hepatozoonoses have been confirmed. Molecular assays have unequivocally confirmed the presence of DNA of E. canis and H. canis in dogs with clinical and pathological features associated with those TBDs. One dog was co-infected with both mentioned pathogens.
Based on the obtained results, it is reasonable to expect the endemic occurrence of CME and canine hepatozoonosis in Serbia. We appeal to veterinary practitioners to include in the differential diagnosis these TBDs in suspicious dogs. Co-infection with different TBPs should also be considered.